Comparison of HPLC Separation of Vitamin D₃ Metabolites and Their Isotachysterol₃ Derivatives

Abstract

Separation of vitamin D₃, its four metabolites and their corresponding isotachysterol₃ derivatives was studied using four HPLC systems: two reverse phase columns, a silica column and a cyanopropyl silica column. Most of them gave a good separation between the compounds studied, although both reverse phase systems were less efficient in the separation of dihydroxylated vitamin D₃ metabolites and isotachysterol compounds. Isotachysterol₃ derivatives behaved analogously to their vitamin D₃ counterparts on all four systems, but their retention times were different. This indicates that chemical derivatization to isotachysterols can be used as a part of chromatographic identification of unknown vitamin D₃ metabolites as well as in the determination of major vitamin D₃ compounds in biological samples.